The search for ‘evolution-proof’ antibiotics

The effectiveness of antibiotics has been widely compromised by the evolution of resistance among pathogenic bacteria. It would be restored by the development of antibiotics to which bacteria cannot evolve resistance. We first discuss two kinds of ‘evolution-proof’ antibiotic. The first comprises literally evolution-proof antibiotics to which bacteria cannot become resistant by mutation or horizontal gene transfer. The second category comprises agents to which resistance may arise, but so rarely that it does not become epidemic. The likelihood that resistance to a novel agent will spread is evaluated here by a simple model that includes biological and therapeutic parameters governing the evolution of resistance within hosts and the transmission of resistant strains between hosts. This model leads to the conclusion that epidemic spread is unlikely if the frequency of mutations that confer resistance falls below a defined minimum value, and it identifies potential targets for intervention to prevent the evolution of resistance. Whether or not evolution-proof antibiotics are ever found, searching for them is likely to improve the deployment of new and existing agents by advancing our understanding of how resistance evolves

Testing the role of multicopy plasmids in the evolution of antibiotic resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This "evolutionary rescue" approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains

Xba I Site Loss Mutants and Deletion/Duplication Variants of Herpes Simplex Virus Type 1: Isolation, Characterization and Recombination Studies

The aim of this project was to isolate a herpes simplex virus type 1 Glasgow strain 17 genome lacking all four Xba I restriction enzyme sites and to use the sites as non-selected markers to study intratypic HSV recombination. However, a large part of the work involved the analysis of variant genomes which were identified during the isolation of Xba I site negative viruses. The parent virus used in these studies was the variant X2, from which the 0.07 map unit (m.u.) and 0.29 m.u. Xba I sites had been removed by selection enrichment (Brown et al., 1984). The remaining two Xba I sites were deleted as follows s the Xba I site at 0.45 m.u. , which lies within the gene (UL 33) encoding a predicted polypeptide of 14,000 molecular weight (mol. wt.), was removed by selection enrichment; while the Xba I site at 0.29 m.u., which lies within the gene (UL22) encoding the essential glycoprotein H (Buckmaster et al., 1984), was removed by site-directed mutagenesis. The variant devoid of Xba I sites (1702) shows normal growth characteristics in vitro and its polypeptide profile is indistinguishable from wild-type virus apart from the absence of the thymidine kinase (tk) polypeptide, a feature which is believed to be unrelated to the loss of the Xba I sites. During in vitro latency experiments, Cook and Brown (1987) isolated a variant of X2 containing a novel Xba I site at 0.74 m.u. This virus was used in intratypic recombination experiments with wild-type strain 17 to generate a HSV-1 strain 17 variant (1708) containing a fifth Xba I site, thus increasing the number of non-selectable markers between this virus (5) and 1702 (0). Different temperature-sensitive (ts) lesions were introduced into 1702 and 1708 as selectable markers. A time-course of recombination was carried out at the permissive temperature and the appearance of non-ts recombinants assayed at the non-pemissive temperature. Recombination was first detected at 4 h post infection, following the onset of DNA replication, and rapidly increased to 15% by 24 h post infection. The distribution of the non-selectable markers, ie. the Xba I sites, in ts+ recombinant molecules was analyzed. From the experimental results a number of conclusions were drawn: (i) there was an increase in the complexity or number of crossovers in recombinants arising at later time-points, confirming the previous hypothesis of Ritchie et al. (1977) that both parental and progeny molecules take part in HSV recombination; (ii) in ts+ recombinants, recombination was very high outwith the selected recombination region, suggesting that correct alignment of genomes plays an important role in determining the overall but not the relative rate of recombination; and (iii) due to the large distance between the Xba I sites, little can be determined about recombinational hotspots. The HSV-1 genome consists of two unique sequences - the long unique (UL) and the short unique (US) - flanked by inverted repeat elements known as the internal repeats (IRL/IRS) and the terminal repeats (TRL/TRS). During the isolation of 1702, a variant (1703) was isolated which has a deletion of approximately 5x10e6 mol. wt. in the UL and IRL regions of its genome, such that one copy of the immediate-early (IE) gene 1 and two unique open reading frames coding for predicted polypeptides of 20,000 mol. wt. and 22,000 mol. wt. (UL55 and UL56) are deleted. The variant 1703 synthesizes reduced levels of VmwIE110, the product of IE gene 1, and under immediate-early conditions apparently fails to synthesize VmwIE63, at both the polypeptide and RNA levels, despite there being no apparent deletion in the coding or controlling regions of the IE2 (VmwIE63) gene. 1703 also fails to synthesize the thymidine kinase polypeptide, although this is unrelated to either the deletion or the failure to synthesize VmwIE63. The variant 1703 exhibits normal growth characteristics in vitro. During the analysis of eighty plaque isolates from one recombination experiment, fourteen variants with rearrangements around the long repeats were detected. Of these, eleven have extensive variation (up to 0.4x10e6 mol. wt.) in the size of the long repeats outwith the 'a' sequence. The remaining three variants have large scale deletion or duplication of both unique and repeat sequences

A Comparative Study Investigating Education and Language Policy in Scotland (With Respect to Gaelic) and Israel (With Respect to Hebrew)

Gaelic education in Scotland has no developed system for second language learning on an intensive, immersive, communicative basis. The Israeli situation was investigated and used as a comparator and contrastor to inform the present revival of the Gaelic language and to suggest possible future developments for intensive immersion language courses. Israel has long experience in second-language teaching and their ulpan method is renowned throughout the world. Both the Basques and the Welsh have adopted ulpan techniques. It was felt that the Gaelic situation could be best informed by investigating the ulpan system at source, and then to take cognisance of how two minority languages, Euskara and Welsh, had implemented an Israeli-based technique. The Irish situation was included to provide a more closely-related comparator with Gaelic. The methodology used in this investigation includes: taped personal interviews; personal learning of immersion techniques by participating in a 24 day Hebrew immersion course in Israel; personal observation of classes in language schools; and desk research. Two visits to Israel were undertaken, one for 12 days (23 December 1991-3 January 1992) and the other for 6 weeks, spanning July and August 1992. The Basque Region of Spain (11-15 February, 1991), the Republic of Ireland (21-25 January 1991,) Wales (25-28 May 1993) and Friesland (20-25 March 1994) have also been visited to inform thinking for this research study. As far as is known, the study is the first to investigate Israeli ulpan methodology for the Gaelic world, and the first to develop formal links with Israeli linguists for this purpose. The main difficulty encountered in this study was the fragmented and limited range of information on immersion methodology

Warming alters life-history traits and competition in a phage community

Host-parasite interactions are highly susceptible to changes in temperature due to mismatches in species thermal responses. In nature, parasites often exist in communities, and responses to temperature are expected to vary between host-parasite pairs. Temperature change thus has consequences for both host-parasite dynamics and parasite-parasite interactions. Here, we investigate the impact of warming (37°C, 40°C, 42°C) on parasite life-history traits and competition using the opportunistic bacterial pathogen Pseudomonas aeruginosa (host) and a panel of three genetically diverse lytic bacteriophages (parasites). We show that phages vary in their responses to temperature; while 37°C and 40°C did not have a major effect on phage infectivity, infection by two phage was restricted at 42°C. This outcome was attributed to disruption of different phage lifehistory traits including host attachment and replication inside hosts. Further, we show that temperature mediates competition between phages by altering their competitiveness. These results highlight phage trait variation across thermal regimes with the potential to drive community dynamics. Our results have important implications for eukaryotic viromes and the design of phage cocktail therapies

Antibiotic resistance alters the ability of Pseudomonas aeruginosa to invade bacteria from the respiratory microbiome

The emergence and spread of antibiotic resistance in bacterial pathogens is a global health threat. One important unanswered question is how antibiotic resistance influences the ability of a pathogen to invade the host-associated microbiome. Here we investigate how antibiotic resistance impacts the ability of a bacterial pathogen to invade bacteria from the microbiome, using the opportunistic bacterial pathogen Pseudomonas aeruginosa and the respiratory microbiome as our model system. We measure the ability of P. aeruginosa spontaneous antibiotic-resistant mutants to invade pre-established cultures of commensal respiratory microbes in an assay that allows us to link specific resistance mutations with changes in invasion ability. While commensal respiratory microbes tend to provide some degree of resistance to P. aeruginosa invasion, antibiotic resistance is a double-edged sword that can either help or hinder the ability of P. aeruginosa to invade. The directionality of this help or hindrance depends on both P. aeruginosa genotype and respiratory microbe identity. Specific resistance mutations in genes involved in multidrug efflux pump regulation are shown to facilitate the invasion of P. aeruginosa into Staphylococcus lugdunensis, yet impair invasion into Rothia mucilaginosa and Staphylococcus epidermidis. Streptococcus species provide the strongest resistance to P. aeruginosa invasion, and this is maintained regardless of antibiotic resistance genotype. Our study demonstrates how the cost of mutations that provide enhanced antibiotic resistance in P. aeruginosa can crucially depend on community context. We suggest that attempts to manipulate the microbiome should focus on promoting the growth of commensals that can increase the fitness costs associated with antibiotic resistance and provide robust inhibition of both wildtype and antibiotic-resistant pathogen strains

Mixed strain pathogen populations accelerate the evolution of antibiotic resistance in patients

Antibiotic resistance poses a global health threat, but the within-host drivers of resistance remain poorly understood. Pathogen populations are often assumed to be clonal within hosts, and resistance is thought to emerge due to selection for de novo variants. Here we show that mixed strain populations are common in the opportunistic pathogen P. aeruginosa. Crucially, resistance evolves rapidly in patients colonized by multiple strains through selection for pre-existing resistant strains. In contrast, resistance evolves sporadically in patients colonized by single strains due to selection for novel resistance mutations. However, strong trade-offs between resistance and growth rate occur in mixed strain populations, suggesting that within-host diversity can also drive the loss of resistance in the absence of antibiotic treatment. In summary, we show that the within-host diversity of pathogen populations plays a key role in shaping the emergence of resistance in response to treatment

Adaptive radiation and the evolution of resource specialization in experimental populations of Pseudomonas fluorescens

Understanding the origins of biological diversity is a fundamental goal of evolutionary biology. A large body of theory attributes ecological and genetic diversification to divergent natural selection for resource specialization. This thesis examines adaptive radiation in response to selection for resource specialization in microcosm populations of the asexual bacterium Pseudomonas fluorescens. The general protocol for these experiments is to introduce a clonal population of Pseudomonas into a novel environment and to allow evolution to occur through the spontaneous appearance of novel genotypes carrying beneficial mutations. Adaptation can then be quantified through direct comparisons between evolved populations and their clonal ancestors. These experiments show that resource heterogeneity generates divergent natural selection for specialization on alternative resources, irrespective of the spatial structure of the environment. Adaptive radiation is possible in sympatry because of genetic trade-offs in the ability to exploit different resources, but these trade-offs are often not the result of antagonistic pleiotropy among loci that determine fitness on alternative resources. The rate of phenotypic diversification declines during adaptive radiation, apparently because the ecological opportunities required to support specialist lineages disappear as a consequence of initial diversification. The ultimate outcome of repeated instances of adaptive radiation is the evolution of a community of ecologically equivalent specialists that share similar adaptive traits, despite differences in the underlying genetic basis of specialization in replicate radiations. Comparisons with the literature on experimental evolution in microbial populations illustrate the results of this thesis are well-supported by experiments in a wide range of microbial microcosms